Abstract/Summary: Alcohol use disorder is a complex group of disorders involving excessive uncontrolled alcohol intake. Our recent human imaging data suggest distinct circuits of interest in male and female AUD subjects during abstinence. In this proposal, we will explore the insula-BNST circuit in female mice, testing whether negative affective disturbances that emerge during abstinence are associated with the relative strength of this pathway. We will further assess the therapeutic potential of the endocannabinoid system at this synapse by utilizing a novel genetic strategy of deletion of the CB1 cannabinoid receptor and the endocannabinoid 2-AG synthetic enzyme DAG lipase in patterns to determine its specific contribution to AUD-related behaviors at the insula- BNST synapse. In male mice, we will explore the disruption of the ventral hippocampal-BNST-hypothalamic circuit suggested by human imaging data in a model of operant alcohol seeking behavior. Finally, we will perform novel tracing strategies to determine afferent inputs to these pathways to inform future human imaging studies.